Resistance to Invading Phages: CRISPR/Cas9 in Bacteria

In genetic engineering many attempts have been made to manipulate genomes of any organism. The ways of genome engineering commonly include homologous recombination, RNA interference (RNAi), Zinc-finger-nucleases. In homologous recombination the efficacy is very low due to the large distribution of similar flanking regions in the genome and success of genome targeting depends on integration of transgene at the site as desired. But possibility is that, due to transposing elements the flanking regions of the genome may transpose to a region where genes goes silent or to a region of over expression, therefore due to this reason the results are not much fruitful. In RNAi the success rate cannot be determined and there are also chances of non-targeted gene silencing. In 2012, a demonstration of a new tool was given; this tool had the ability to knock off the genes at specific sites. This genome editing tool was the major breakthrough in the science and it was reported in 2007 by Barrangou and colleagues, it is called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated-9 protein nucleases (Cas9). They were known in 1980s but their major function of genome editing remained a mystery until 2012. They have the role in bacterial immunity.

Mechanism of Action:

There are three different types of CRIPSPR/Cas systems out of which 2nd type is of most importance. The function of type 2 of CRISPR and Cas9 proteins is to provide immunity against invading phages to Bacteria and Achaea. This CRISPR/Cas9 system works in 3 phases to provide immunity. Firstly, on the invasion of phage, CRISPR-associated 1 and 2 protein nucleases (Cas 1 and Cas2) recognize a specific target region in the phage genome cuts phage genome into small pieces and incorporate those pieces within the short repeats of CRISPR to remember invading phage for future prevention. After that, CRISPR genes are transcribed into pre-CRISPR-RNA (crRNA), this pre-crRNA is cleaved by endonucleases at repeating sequences and now the mature-crRNA is formed and endonucleases remain attached to the mature crRNA forming crRNA-endonuclease complex. This crRNA-endonuclease complex then binds with other Cas protein nucleases and in the last this complex of crRNA-endonucleases then finds the complementary DNA sequences in the phage genome that invades the bacterium in the future and binds to that region. This crRNA-endonuclease complex then recruits a helicase-nuclease called Cas-3 which chops down the invading DNA.

Cas9 Adoption in Molecular Biology:

Up to now 3 different types of CRIPSPR/Cas systems have been adopted for genome editing in Molecular Biology which are:

The wild type Cas9 which cleaves the invading phage DNA at specific sites and then activates a double stranded break-repair mechanism as a result of which the chances of non-homologous insertions and deletions increases and the invading phage DNA becomes inactive, but if we allow homologous repairing by providing target locus then desired mutations could be achieved.

Type 2, the most widely studied type is actually a mutant created by Cong and colleagues known as Cas9D10A. This mutant only has the cutting activity and devoid of break-repair activity, it can cut only single strand of DNA. When provided a homologous repair template, then the repair occurs with great precision without any insertion or deletion leading to mutagenesis. It is actually designed to generate adjacent nicks in DNA.

Third one is nuclease-deficient Cas9 (dCas9). This system is used to target genomes without any cleavage. This type could be used either as silencing or gene activating tool.

Applications:

This CRISPR Cas9 system allowed the targeted genome engineering with more effective percentage, e.g. in humans using Zinc-Finger-Nucleases the target genome engineering efficiency achieved was only 1-50%, but with Cas9 system this efficiency in humans was more than 70% which showed that CRISPR/Cas9 system has the highest efficacy in genome engineering, and not only in humans this Cas9 system has also been successfully used in genome targeting of plants, Drosophila, zebrafish, C. elegans, rabbits, monkeys and yeast etc. and with more efforts and researches in this field, it would open up more ways of genome editing. Recently the dCas9 system is being used for targeting protein domains to induce transcriptional regulation, epigenetic modifications and to induce point mutations and visualizing genome loci etc.